Summary of Work: The flavin-containing monooxygenase gene family contains five known members. These drug metabolizing enzymes have wide, overlapping substrate specificities and participate in the oxidative metabolism of numerous drugs, pesticides and other environmental chemicals. Of these enzymes, we have shown that FMO3 is the most important with respect to drug metabolism in human liver. In addition to FMO3, both FMO4 and FMO5 are expressed in human liver. The properties of FMO5 have been established, but those of FMO4 have been difficult to assess because of difficulty in expressing this isoform in E. coli. Recently we expressed a modified (truncated) FMO3 and have now identified the region of the full-length cDNA that appears to block translation. A sequence of five nucleotides located 57 bases to the 5' of the stop codon forms a complex with a sequence located 42 bases from the start codon. Introduction of silent mutations in either the 3' or 5' region of the complex results in successful expression in E. coli. The interaction of tricyclic antidepressants with the FMO1 has been studied with chimeras constructed from the rabbit and pig FMO1 orthologs and by site-directed mutagenesis. Introduction of pig amino acids at five positions in the rabbit protein completely changes the properties of the rabbit isoform to those of the pig isoform. One of these positions, 433, appears to be critical in determining whether FMO2 is activated or inhibited by the antidepressants. FMO3 has also been shown to interact with chemical modulators with results dependent upon substrate concentrations; activation at high concentrations and inhibition at low concentrations. These results, which were obtained with recombinant human FMO3, are being examined in human liver microsomal samples.